Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

The Impact of COVID-19 on Multidrug-Resistant Bacteria at a Slovenian Tertiary Medical Center.

The COVID-19 pandemic has strained healthcare systems globally. Shortages of hospital beds, reassignment of healthcare workers to COVID-19-dedicated wards, an increased workload, and evolving infection prevention and control measures have potentially contributed to the spread of multidrug-resistant bacteria (MDRB). To determine the impact of the COVID-19 pandemic at the University Medical Center Ljubljana, a tertiary teaching hospital, we analyzed the monthly incidence of select bacterial species per patient from 2018 to 2022. The analysis was performed for all isolates and for MDRB isolates. The data were analyzed separately for isolates from all clinical samples, from blood culture only, and from clinical and surveillance samples. Our findings revealed an increased incidence density of patients with Enterococcus faecium, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa isolates from clinical samples during the COVID-19 period in the studied hospital. Notably, the incidence density of MDRB isolates-vancomycin-resistant E. faecium, extended-spectrum betalactamase-producing K. pneumoniae, and betalactam-resistant P. aeruginosa-from clinical samples increased during the COVID-19 period. There were no statistically significant differences in the incidence density of patients with blood culture MDRB isolates. We observed an increase in the overall MDRB burden (patients with MDRB isolates from both clinical and surveillance samples per 1000 patient days) in the COVID-19 period in the studied hospital for vancomycin-resistant E. faecium, carbapenem-resistant K. pneumoniae, and betalactam-resistant P. aeruginosa and a decrease in the methicillin-resistant S. aureus burden.

Prospective Longitudinal Study of Dynamics of Human Papillomavirus 6 and 11 Infection in Anogenital Hairs and Eyebrows of Male Patients with Anogenital Warts and Age-Matched Controls.

To better understand the natural history of anogenital warts (AGWs) and the dynamics of HPV6/11 infection in regional hairs, 32 newly diagnosed male patients with AGWs and 32 age-matched healthy controls were closely followed. During enrollment and six follow-up visits (every 2.6 months), 43 AGW tissues and 1232 anogenital and eyebrow hair samples were collected. This is the closest longitudinal monitoring of AGW patients to date. Patients were treated according to standards of care. The HPV6/11 prevalence was 19.9% in the patients' hair samples (HPV6 B1 in 53.1%) and 0% in the controls. The highest HPV6/11 prevalence was found in pubic hairs (29.0%) and the lowest in eyebrows (7.1%). The odds of having HPV6/11-positive hairs increased with smoking, shaving the anogenital region, and age. A close association between HPV6/11 presence in hairs and clinically visible AGWs was observed. The proportion of patients with visible AGWs and HPV6/11-positive hairs declined during follow-up with similar trends. No particular HPV6/11 variant was linked with an increased AGW recurrence, but the sublineage HPV6 B1 showed significantly higher clearance from hairs. Despite treatment, 78.1% and 62.5% of the AGW patients experienced one and two or more post-initial AGW episodes, respectively. The patients with HPV6/11-positive hairs or visible AGWs at a preceding visit demonstrated substantially higher odds of presenting with visible AGWs at a subsequent visit.

Molecular characterization of extended-spectrum ß-lactamase-producing Escherichia coli isolated from lower respiratory tract samples between 2002 and 2019 in the Central Slovenia region.

BACKGROUND: Antibiotic resistance is one of the most serious global health problems and threatens the effective treatment of bacterial infections. Of greatest concern are infections caused by extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC). The aim of our study was to evaluate the prevalence and molecular characteristics of ESBL-EC isolated over an 18-year pre-COVID period from lower respiratory tract (LRT) samples collected from selected Slovenian hospitals. OBJECTIVES AND METHODS: All isolates were identified by MALDI-TOF and phenotypically confirmed as ESBLs by a disk diffusion assay. Using a PCR approach, 487 non-repetitive isolates were assigned to phylogroups, sequence type groups, and clonal groups. Isolates were also screened for virulence-associated genes (VAGs) and antimicrobial resistance genes. RESULTS: The prevalence of ESBL-EC isolates from LRT in a large university hospital was low (1.4%) in 2005 and increased to 10.8% by 2019. The resistance profile of 487 non-repetitive isolates included in the study showed a high frequency of group 1 blaCTX-M (77.4%; n = 377), blaTEM (54.4%; n = 265) and aac(6')-Ib-cr (52%; n = 253) genes and a low proportion of blaSHV and qnr genes. Isolates were predominantly assigned to phylogroup B2 (73.1%; n = 356), which was significantly associated with clonal group ST131. The ST131 group accounted for 67.6% (n = 329) of all isolates and had a higher number of virulence factor genes than the non-ST131 group. The virulence gene profile of ST131 was consistent with that of other extraintestinal pathogenic E. coli (ExPEC) strains and was significantly associated with ten of sixteen virulence factor genes tested. Using ERIC-PCR fingerprinting, isolates with the same ERIC-profile in samples from different patients, and at different locations and sampling dates were confirmed, indicating the presence of "hospital-adapted" strains. CONCLUSION: Our results suggest that the ESBL-EC isolates from LRT do not represent a specific pathotype, but rather resemble other ExPEC isolates, and may be adapted to the hospital environment. To our knowledge, this is the first study of ESBL-EC isolated from LRT samples collected over a long period of time.

QAC Resistance Genes in ESBL-Producing E. coli Isolated from Patients with Lower Respiratory Tract Infections in the Central Slovenia Region-A 21-Year Survey.

Biocidal products prevent the spread of pathogenic microorganisms, including extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC), which is one of the most alarming health problems worldwide. Quaternary ammonium compounds (QACs) are surface-active agents that interact with the cytoplasmic membrane and are widely used in hospitals and food processing environments. A collection of 577 ESBL-EC, isolated from lower respiratory tract (LRT) samples, was screened for QAC resistance genes oqxA; oqxB; qacEΔ1; qacE; qacF/H/I; qacG; sugE (p); emrE; mdfA; sugE (c); ydgE; ydgF; and for class 1, 2, and 3 integrons. The prevalence of chromosome-encoded genes ranged from 77 to 100%, while the prevalence of QAC resistance genes encoded on mobile genetic elements (MGEs) was relatively low (0-0.9%), with the exception of qacEΔ1 (54.6%). PCR screening detected the presence of class 1 integrons in 36.3% (n = 210) of isolates, which were positively correlated with qacEΔ1. More correlations between QAC resistance genes, integrons, sequence type group ST131, and ß-lactamase genes were presented. The results of our study confirm the presence of QAC resistance genes and also class 1 integrons commonly found in multidrug-resistant clinical isolates and highlight the potential role of QAC resistance genes in the selection of ESBL-producing E. coli in hospitals.

Ceramide Phosphoethanolamine as a Possible Marker of Periodontal Disease.

Periodontal disease is a chronic oral inflammatory disorder initiated by pathobiontic bacteria found in dental plaques-complex biofilms on the tooth surface. The disease begins as an acute local inflammation of the gingival tissue (gingivitis) and can progress to periodontitis, which eventually leads to the formation of periodontal pockets and ultimately results in tooth loss. The main problem in periodontology is that the diagnosis is based on the assessment of the already obvious tissue damage. Therefore, it is necessary to improve the current diagnostics used to assess periodontal disease. Using lipidomic analyses, we show that both crucial periodontal pathogens, Porphyromonas gingivalis and Tannerella forsythia, synthesize ceramide phosphoethanolamine (CPE) species, membrane sphingolipids not typically found in vertebrates. Previously, it was shown that this particular lipid can be specifically detected by an aegerolysin protein, erylysin A (EryA). Here, we show that EryA can specifically bind to CPE species from the total lipid extract from P. gingivalis. Furthermore, using a fluorescently labelled EryA-mCherry, we were able to detect CPE species in clinical samples of dental plaque from periodontal patients. These results demonstrate the potential of specific periodontal pathogen-derived lipids as biomarkers for periodontal disease and other chronic inflammatory diseases.

The effects of antibiotic cycling and mixing on acquisition of antibiotic resistant bacteria in the ICU: A post-hoc individual patient analysis of a prospective cluster-randomized crossover study.

BACKGROUND: Repeated rotation of empiric antibiotic treatment strategies is hypothesized to reduce antibiotic resistance. Clinical rotation studies failed to change unit-wide prevalence of antibiotic resistant bacteria (ARB) carriage, including an international cluster-randomized crossover study. Unit-wide effects may differ from individual effects due to "ecological fallacy". This post-hoc analysis of a cluster-randomized crossover study assesses differences between cycling and mixing rotation strategies in acquisition of carriage with Gram-negative ARB in individual patients. METHODS: This was a controlled cluster-randomized crossover study in 7 ICUs in 5 European countries. Clinical cultures taken as routine care were used for endpoint assessment. Patients with a first negative culture and at least one culture collected in total were included. Community acquisitions (2 days of admission or less) were excluded. Primary outcome was ICU-acquisition of Enterobacterales species with reduced susceptibility to: third- or fourth generation cephalosporins or piperacillin-tazobactam, and Acinetobacter species and Pseudomonas aeruginosa with reduced susceptibility for piperacillin-tazobactam or carbapenems. Cycling (altering first-line empiric therapy for Gram-negative bacteria, every other 6-weeks), to mixing (changing antibiotic type every empiric antibiotic course). Rotated antibiotics were third- or fourth generation cephalosporins, piperacillin-tazobactam and carbapenems. RESULTS: For this analysis 1,613 admissions were eligible (855 and 758 during cycling and mixing, respectively), with 16,437 microbiological cultures obtained. Incidences of acquisition with ARB during ICU-stay were 7.3% (n = 62) and 5.1% (n = 39) during cycling and mixing, respectively (p-value 0.13), after a mean of 17.7 (median 15) and 20.8 (median 13) days. Adjusted odds ratio for acquisition of ARB carriage during mixing was 0.62 (95% CI 0.38 to 1.00). Acquired carriage with ARB were Enterobacterales species (n = 61), Pseudomonas aeruginosa (n = 38) and Acinetobacter species (n = 20), with no statistically significant differences between interventions. CONCLUSIONS: There was no statistically significant difference in individual patients' risk of acquiring carriage with Gram-negative ARB during cycling and mixing. These findings substantiate the absence of difference between cycling and mixing on the epidemiology of Gram-negative ARB in ICU. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, registered 10 January 2011, NCT01293071.

Collaborative Robot Precision Task in Medical Microbiology Laboratory.

This study focuses on the feasibility of collaborative robot implementation in a medical microbiology laboratory by demonstrating fine tasks using kinesthetic teaching. Fine tasks require sub-millimetre positioning accuracy. Bacterial colony picking and identification was used as a case study. Colonies were picked from Petri dishes and identified using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. We picked and identified 56 colonies (36 colonies of Gram-negative Acinetobacter baumannii and 20 colonies of Gram-positive Staphylococcus epidermidis). The overall identification error rate was around 11%, although it was significantly lower for Gram-positive bacteria (5%) than Gram-negative bacteria (13.9%). Based on the identification scores, it was concluded that the system works similarly well as a manual operator. It was determined that tasks were successfully demonstrated using kinesthetic teaching and generalized using dynamic movement primitives (DMP). Further improvement of the identification error rate is possible by choosing a different deposited sample treatment method (e.g., semi-extraction, wet deposition).

European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines for the treatment of infections caused by multidrug-resistant Gram-negative bacilli (endorsed by European society of intensive care medicine).

SCOPE: These ESCMID guidelines address the targeted antibiotic treatment of third-generation cephalosporin-resistant Enterobacterales (3GCephRE) and carbapenem-resistant Gram-negative bacteria, focusing on the effectiveness of individual antibiotics and on combination versus monotherapy. METHODS: An expert panel was convened by ESCMID. A systematic review was performed including randomized controlled trials and observational studies, examining different antibiotic treatment regimens for the targeted treatment of infections caused by the 3GCephRE, carbapenem-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii. Treatments were classified as head-to-head comparisons between individual antibiotics and between monotherapy and combination therapy regimens, including defined monotherapy and combination regimens only. The primary outcome was all-cause mortality, preferably at 30 days and secondary outcomes included clinical failure, microbiological failure, development of resistance, relapse/recurrence, adverse events and length of hospital stay. The last search of all databases was conducted in December 2019, followed by a focused search for relevant studies up until ECCMID 2021. Data were summarized narratively. The certainty of the evidence for each comparison between antibiotics and between monotherapy and combination therapy regimens was classified by the GRADE recommendations. The strength of the recommendations for or against treatments was classified as strong or conditional (weak). RECOMMENDATIONS: The guideline panel reviewed the evidence per pathogen, preferably per site of infection, critically appraising the existing studies. Many of the comparisons were addressed in small observational studies at high risk of bias only. Notably, there was very little evidence on the effects of the new, recently approved, ß-lactam/ß-lactamase inhibitors on infections caused by carbapenem-resistant Gram-negative bacteria. Most recommendations are based on very-low- and low-certainty evidence. A high value was placed on antibiotic stewardship considerations in all recommendations, searching for carbapenem-sparing options for 3GCephRE and limiting the recommendations of the new antibiotics for severe infections, as defined by the sepsis-3 criteria. Research needs are addressed.

A retrospective analysis of clinical characteristics and management of perianal streptococcal dermatitis in children and adults.

INTRODUCTION: Due to the paucity of recent literature on perianal streptococcal disease (PSD), we performed a comprehensive analysis of clinical characteristics of PSD and its management. METHODS: We conducted a retrospective search in the laboratory information system of the Institute of Microbiology and Immunology, Ljubljana, Slovenia, between January 2006 and December 2016 and identified patients with suspected PSD. We reviewed patients' medical records and obtained data on patient age and sex, concomitant illnesses, duration of complaints, signs and symptoms of PSD, epidemiological history, date of diagnosis, microbiological characteristics of beta-hemolytic streptococcal isolates, additional laboratory findings, duration and type of systemic and/or topical therapy, and recurrence of PSD. RESULTS: We identified 64 pediatric and eight adult PSD cases in total. The most common signs and symptoms were perianal erythema (67/72; 93.1%), anal fissures (28/72; 38.8%), itching (22/72; 30.6%), and blood-streaked stools (19/72; 26.4%). The duration of symptoms varied from < 1 week to > 1 year, with 58.3% of patients experiencing symptoms between 1 week and 6 months. The majority of patients received systemic (63/72; 87.5%) and topical (56/72; 77.8%) treatment. CONCLUSIONS: Although the signs and symptoms of PSD are non-specific, clinicians should be highly suspicious of the disease in adults and especially in preschool children with perianal complaints. Despite being a common disease, there is still considerable delay in correct diagnosis and treatment, prolonging the discomfort of PSD patients.

Molecular Characterization of Human Papillomavirus Type 159 (HPV159).

Human papillomavirus type 159 (HPV159) was identified in an anal swab sample and preliminarily genetically characterized by our group in 2012. Here we present a detailed molecular in silico analysis that showed that the HPV159 viral genome is 7443 bp in length and divided into five early and two late genes, with conserved functional domains and motifs, and a non-coding long control region (LCR) with significant regulatory sequences that allow the virus to complete its life cycle and infect novel host cells. HPV159, clustering into the cutaneotropic Betapapillomavirus (Beta-PV) genus, is phylogenetically most similar to HPV9, forming an individual phylogenetic group in the viral species Beta-2. After testing a large representative collection of clinical samples with HPV159 type-specific RT-PCR, in addition to the anal canal from which the first HPV159 isolate was obtained, HPV159 was further detected in other muco-cutaneous (4/181, 2.2%), mucosal (22/764, 2.9%), and cutaneous (14/554, 2.5%) clinical samples, suggesting its extensive tissue tropism. However, because very low HPV159 viral loads were estimated in the majority of positive samples, it seemed that HPV159 mainly caused clinically insignificant infections of the skin and mucosa. Using newly developed, highly sensitive HPV159-specific nested PCRs, two additional HPV159 LCR viral variants were identified. Nevertheless, all HPV159 mutations were demonstrated outside important functional domains of the LCR, suggesting that the HPV159 viral variants were most probably not pathogenically different. This complete molecular characterization of HPV159 enhances our knowledge of the genome characteristics, tissue tropism, and phylogenetic diversity of Beta-PVs that infect humans.

Feline Otitis Externa Caused by Methicillin-Resistant Staphylococcus aureus with Mixed Hemolytic Phenotype and Overview of Possible Genetic Backgrounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in humans, but its importance in small animal practice is increasing. Here, we present a case of feline otitis externa (OE) caused by MRSA; both hemolytic and nonhemolytic variants with a stable phenotype were recovered from the external auditory canal after infection was detected by routine otoscopy. One isolate per variant underwent antimicrobial susceptibility testing (AST) by broth microdilution method, conventional spa typing and whole-genome sequencing (WGS). The results showed that both variants were genetically related and were of sequence type (ST) 1327, SCCmec type IV and spa type t005. AST and WGS showed that both isolates were resistant to ß-lactams and sensitive to all tested non-ß-lactam antibiotics. Both isolates were pvl-negative, but encoded several other virulence genes (aur, hlgABC, sak, scn, seg, sei, sem, sen, seo and seu). Genetic background of the mixed hemolytic phenotype was not identified; no differences in the agr locus or other regulatory regions were detected. Three single-nucleotide polymorphisms were identified but could not be associated with hemolysis. This well-documented case of MRSA infection in companion animals adds to the reports of MRSA infections with a mixed hemolytic phenotype.

The genetic diversity of human papillomavirus types from the species Gammapapillomavirus 15: HPV135, HPV146, and HPV179.

OBJECTIVES: To determine the prevalence, viral load, tissue tropism, and genetic variability of novel human papillomavirus (HPV) type 179, which is etiologically associated with sporadic cases of common warts in immunocompromised patients, and phylogenetically related HPV types 135 and 146. METHODS: The representative collection of 850 HPV-associated clinical samples (oral/nasopharyngeal/anal, archival specimens of oral/oropharyngeal/conjunctival/cervical/skin cancer, benign lesions of the larynx/conjunctiva/skin, and eyebrows), obtained from immunocompetent individuals, was tested for the presence of HPV179, HPV135, and HPV146 using type-specific real-time PCRs. To assess the genetic diversity of the HPVs investigated in the non-coding long control region (LCR), several highly sensitive nested PCR protocols were developed for each HPV type. The genetic diversity of HPV179 was additionally determined in 12 HPV179 isolates from different anatomical sites of an only immunocompromised patient included in the study. RESULTS: HPV179, HPV135, and HPV146 were detected in 1.4, 2.0, and 1.5% of the samples tested, respectively, with no preference for cutaneous or mucosal epithelial cells. One (with five single nucleotide polymorphisms; SNPs), four (with one to six SNPs), and four (with one to eight SNPs) genetic variants of HPV179, HPV135, and HPV146, respectively, were identified among eligible samples. HPV179 isolates from the immunocompromised patient exhibited the identical LCR nucleotide sequence, suggesting that HPV179 can cause generalized HPV infections. CONCLUSIONS: HPV179, HPV135, and HPV146 have a mucocutaneous tissue tropism and are associated with sporadic infections in immunocompromised and immunocompetent individuals. Because the majority of mutations were found outside the major functional domains of the respective LCRs, we assume that HPV179, HPV135, and HPV146 genetic variants pathogenically do not differ from their prototypes. In addition, no association was found between specific HPV179, HPV135, and HPV146 genetic variants and anatomical sites of infection and/or specific neoplasms.

Real-Life Head-to-Head Comparison of Performance of Two High-Throughput Automated Assays for the Detection of SARS-CoV-2 RNA in Nasopharyngeal Swabs: The Alinity m and cobas 6800 SARS-CoV-2 Assays.

The Alinity m (Abbott Molecular, Des Plaines, IL) automated molecular analyzer allows continuous loading of samples and sample-to-result molecular detection of several microorganisms. The detection of SARS-CoV-2 by the Alinity m was compared with that of the cobas 6800 (Roche Molecular Systems, Branchburg, NJ; standard comparator) in a manufacturer-independent clinical evaluation on 2157 consecutive nasopharyngeal swab samples. Valid initial results on Alinity m and cobas 6800 were obtained from 2129 (98.7%) and 2157 (100%) samples, respectively. The overall percent agreement (95% CI) was 98.3% (2092/2129 [97.6%-98.7%]); positive percent agreement, 100% (961/961 [99.6%-100%]); negative percent agreement, 96.8% (1131/1168 [95.7%-97.7%]); and high κ value, 0.965 (0.954-0.976). There were 37 discordant results on Alinity m and, based on discordant analyses, including previous and/or follow-up PCR results, 22 could be considered analytically true positive with high probability. Due to a lack of additional information and an inability to perform repeated/further testing, the status of the remaining 15 discordant results remained unresolved. The throughput of the two analyzers was compared using testing on 564 samples in parallel across two 8-hour shifts in clinical practice. The turnaround times were compared using processing of 94 routine samples in parallel on each working day for 5 consecutive days. The two analyzers showed similar performance, with certain differences that have potential importance in some laboratory settings.

Seroprevalence of severe acute respiratory syndrome coronavirus 2 in Slovenia: results of two rounds of a nationwide population study on a probability-based sample, challenges and lessons learned.

OBJECTIVES: Seroprevalence surveys provide crucial information on cumulative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure. This Slovenian nationwide population study is the first longitudinal 6-month serosurvey using probability-based samples across all age categories. METHODS: Each participant supplied two blood samples: 1316 samples in April 2020 (first round) and 1211 in October/November 2020 (second round). The first-round sera were tested using Euroimmun Anti-SARS-CoV-2 ELISA IgG (ELISA) and, because of uncertain estimates, were retested using Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S). The second-round sera were concomitantly tested using Elecsys-N/Elecsys-S. RESULTS: The populations of both rounds matched the overall population (n = 3000), with minor settlement type and age differences. The first-round seroprevalence corrected for the ELISA manufacturer's specificity was 2.78% (95% highest density interval [HDI] 1.81%-3.80%), corrected using pooled ELISA specificity calculated from published data 0.93% (95% CI 0.00%-2.65%), and based on Elecsys-N/Elecsys-S results 0.87% (95% HDI 0.40%-1.38%). The second-round unadjusted lower limit of seroprevalence on 11 November 2020 was 4.06% (95% HDI 2.97%-5.16%) and on 3 October 2020, unadjusted upper limit was 4.29% (95% HDI 3.18%-5.47%). CONCLUSIONS: SARS-CoV-2 seroprevalence in Slovenia increased four-fold from late April to October/November 2020, mainly due to a devastating second wave. Significant logistic/methodological challenges accompanied both rounds. The main lessons learned were a need for caution when relying on manufacturer-generated assay evaluation data, the importance of multiple manufacturer-independent assay performance assessments, the need for concomitant use of highly-specific serological assays targeting different SARS-CoV-2 proteins in serosurveys conducted in low-prevalence settings or during epidemic exponential growth and the usefulness of a Bayesian approach for overcoming complex methodological challenges.

Systemic azithromycin as an adjunct to scaling and root planing in patients with stage III/IV periodontitis: 12-month results of a randomized controlled clinical trial.

OBJECTIVES: To determine whether azithromycin (AZI) as an adjunct to scaling and root planing (SRP), when compared to placebo, decreases the number of sites demonstrating pocket depth (PD) ≥ 5 mm and bleeding on probing (BOP) 12 months post-treatment in stage III/IV periodontitis patients. MATERIALS AND METHODS: In a double-blind randomized parallel-arm placebo-controlled trial, 40 stage III/IV periodontitis patients received steps 1 and 2 of periodontal treatment in two sessions within 7 days. Patients then received systemic antibiotic therapy (n = 20; AZI 500 mg/day, 3 days) or placebo (n = 20). Additional instrumentation of residual diseased sites (DS) - sites with PD ≥ 5 mm and BOP - was performed at the 3-, 6- and 9-month follow-ups. The primary outcome variable was the number of DS at the 12-month re-evaluation. Using a multivariate multilevel logistic regression model, the effects of gender, age, antibiotic therapy, presence of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans, smoking, tooth being a molar and interdental location were evaluated. RESULTS: The number of DS after 12 months was similar in the test (median (Me) = 4, interquartile range (IQR) = 0-6) and control (Me = 3, IQR = 1-6.5) groups. Both groups showed substantial but equivalent improvements in periodontal parameters, with no intergroup differences at initially shallow or deep sites. The logistic regression showed a lower odds ratio (OR) for the healing of DS on molars (OR = 0.29; p < 0.001) and in smokers (OR = 0.36; p = 0.048). CONCLUSION: Stage III/IV periodontitis patients showed significant but comparable improvements in periodontal parameters and the number of residual DS at the 12-month revaluation regardless of treatment type. This may have been the result of the additional instrumentation received by patients at residual DS in both treatment groups. CLINICAL RELEVANCE: Treatment with AZI + SRP provided no additional benefits after 12 months in terms of periodontal parameters or the number of persisting sites with PD ≥ 5 mm + BOP as compared to SRP plus placebo. TRIAL REGISTRATION: EUDRA-CT: 2015-004306-42; https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-004306-42/SI , registered 17. 12. 2015.

Head-to-head comparison of two rapid high-throughput automated electrochemiluminescence immunoassays targeting total antibodies to the SARS-CoV-2 nucleoprotein and spike protein receptor binding domain.

BACKGROUND: Accurate anti-SARS-CoV-2 assays are needed to inform diagnostic, therapeutic, and public health decisions. The first manufacturer-independent head-to-head comparison of two rapid high-throughput automated electrochemiluminescence double-antigen sandwich immunoassays targeting total anti-SARS-CoV-2 antibodies against two different viral proteins, Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S) (Roche Diagnostics), was performed in a routine setting during the exponential growth phase of the epidemic's second wave. METHODS: The diagnostic specificity of Elecsys-N and Elecsys-S was initially evaluated on a panel of 572 pre-COVID-19 samples, showing 100 % specificity of both assays. Elecsys-N/Elecsys-S head-to-head comparison used 3,416 consecutive blood samples from individuals that were tested for the presence of anti-SARS-CoV-2 within commercial out-of-pocket serologic testing. RESULTS: Elecsys-N/Elecsys-S head-to-head comparison showed overall agreement of 98.68 % (3,371/3,416; 95 % CI, 98.23-99.03 %), positive agreement of 95.16 % (884/929; 95 % CI, 93.52-96.41 %), and a high kappa value of 0.996 (95 % CI, 0.956-0.976). Previous SARS-CoV-2 PCR positivity was identified in 14/24 (58.3 %) Elecsys-N negative/Elecsys-S positive individuals and in 4/21 (19.0 %) Elecsys-N positive/Elecsys-S negative individuals. CONCLUSION: The first Elecsys-N/Elecsys-S head-to-head comparison showed excellent agreement of two highly specific and rapid high-throughput automated anti-SARS-CoV-2 assays. An important question is whether laboratories offering two different antibody assays could benefit from combining the assays; if so, should use be concomitant or sequential-and, in the latter case, in which order? Based on our results, we favor concomitant over sequential Elecsys-N/Elecsys-S use when testing individuals for anti-SARS-CoV-2 antibodies in high-incidence settings; for example, during the exponential or stationary growth phase of the COVID-19 epidemic.

The Antibacterial Activity of Human Amniotic Membrane against Multidrug-Resistant Bacteria Associated with Urinary Tract Infections: New Insights from Normal and Cancerous Urothelial Models.

Urinary tract infections (UTIs) represent a serious global health issue, especially due to emerging multidrug-resistant UTI-causing bacteria. Recently, we showed that the human amniotic membrane (hAM) could be a candidate for treatments and prevention of UPEC and Staphylococcus aureus infections. However, its role against multidrug-resistant bacteria, namely methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae, vancomycin-resistant Enterococci (VRE), carbapenem-resistant Acinetobacter baumannii, and Pseudomonas aeruginosa has not yet been thoroughly explored. Here, we demonstrate for the first time that the hAM homogenate had antibacterial activity against 7 out of 11 tested multidrug-resistant strains, the greatest effect was on MRSA. Using novel approaches, its activity against MRSA was further evaluated in a complex microenvironment of normal and cancerous urinary bladder urothelia. Even short-term incubation in hAM homogenate significantly decreased the number of bacteria in MRSA-infected urothelial models, while it did not affect the viability, number, and ultrastructure of urothelial cells. The hAM patches had no antibacterial activity against any of the tested strains, which further exposes the importance of the hAM preparation. Our study substantially contributes to basic knowledge on the antibacterial activity of hAM and reveals its potential to be used as an antibacterial agent against multidrug-resistant bacteria.

Effect of Full-mouth Disinfection Protocol on Glycaemic Control and Subgingival Microbiota in Patients with Type 1 and Type 2 Diabetes.

PURPOSE: To evaluate the effect of a full-mouth disinfection protocol (FMD) on periodontal parameters, glycaemic control and subgingival microbiota of periodontal patients with type 1 and type 2 diabetes, as well as those without diabetes. MATERIALS AND METHODS: This study included 33 patients with periodontitis. Eleven of them were type 1 diabetes patients, 11 were type 2 diabetes patients, and 11 were non-diabetics. At baseline and 3 months after the FMD, the periodontal parameters of each patient were recorded, samples of capillary blood for the chairside assessment of HbA1c were taken, and plaque samples from the two deepest periodontal pockets were collected to test for the presence of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf) and Treponema denticola (Td). RESULTS: Bleeding on probing (BOP), probing pocket depth (PPD), clinical attachment level (CAL) and glycated haemoglobin (HbA1c) decreased statistically significantly (p < 0.05) in all three groups 3 months after FMD. Only the proportion of Pg in the control group decreased statistically significantly (p < 0.05), while the proportion of other bacteria decreased or remained the same, whereby the differences were not statistically significant. Moreover, the proportion of Aa in type 1 diabetics increased statistically significantly (p < 0.05). CONCLUSION: The FMD protocol improves periodontal parameters and glycaemic control of type 1 and type 2 diabetes patients with periodontitis.

Evaluation of two rapid phenotypical tests-Alifax rapid AST colistin test and Rapid Polymyxin NP test-for detection of colistin resistance in Enterobacterales.

Our study evaluates the performance of two rapid phenotypical tests to detect colistin resistance in Enterobacterales: Alifax rapid AST colistin test using the HB&L system and Rapid Polymyxin NP test prepared in-house. A collection of well-characterized 53 colistin-susceptible and 66 colistin-resistantEnterobacterales isolates was used. The results obtained using both rapid tests were compared to the reference broth microdilution. Overall categorical agreement was 81.5% for Alifax test and 98.3% for Rapid Polymyxin NP test. Based on our results, the Rapid Polymyxin NP test is superior to the Alifax test that performed inadequate for Enterobacter spp.